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Canine Adenovirus Downstream Processing Protocol

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Adenovirus

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1089))

Abstract

Adenovirus vectors are efficient gene delivery tools. A major caveat with vectors derived from common human adenovirus serotypes is that most adults are likely to have been exposed to the wild-type virus and exhibit active immunity against the vectors. This preexisting immunity limits their clinical success. Strategies to circumvent this problem include the use of nonhuman adenovirus vectors. Vectors derived from canine adenovirus type 2 (CAV-2) are among the best-studied representatives. CAV-2 vectors are particularly attractive for the treatment of neurodegenerative disorders. In addition, CAV-2 vectors have shown great promise as oncolytic agents in virotherapy approaches and as vectors for recombinant vaccines. The rising interest in CAV-2 vectors calls for the development of scalable GMP compliant production and purification strategies. A detailed protocol describing a complete scalable downstream processing strategy for CAV-2 vectors is reported here. Clarification of CAV-2 particles is achieved by microfiltration. CAV-2 particles are subsequently concentrated and partially purified by ultrafiltration–diafiltration. A Benzonase® digestion step is carried out between ultrafiltration and diafiltration operations to eliminate contaminating nucleic acids. Chromatography purification is accomplished in two consecutive steps. CAV-2 particles are first captured and concentrated on a propyl hydrophobic interaction chromatography column followed by a polishing step using DEAE anion exchange monoliths. Using this protocol, high-quality CAV-2 vector preparations containing low levels of contamination with empty viral capsids and other inactive vector forms are typically obtained. The complete process yield was estimated to be 38–45 %.

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References

  1. Sprangers MC, Lakhai W, Koudstaal W, Verhoeven M, Koel BF, Vogels R et al (2003) Quantifying adenovirus-neutralizing antibodies by luciferase transgene detection: addressing preexisting immunity to vaccine and gene therapy vectors. J Clin Microbiol 41:5046–5052

    Article  PubMed  CAS  Google Scholar 

  2. Aste-Amezaga M, Bett AJ, Wang F, Casimiro DR, Antonello JM, Patel DK et al (2004) Quantitative adenovirus neutralization assays based on the secreted alkaline phosphatase reporter gene: application in epidemiologic studies and in the design of adenovector vaccines. Hum Gene Ther 15:293–304

    Article  PubMed  CAS  Google Scholar 

  3. Bru T, Salinas S, Kremer EJ (2010) An update on canine adenovirus type 2 and its vectors. Viruses 2:2134–2153

    Article  PubMed  CAS  Google Scholar 

  4. Soudais C, Laplace-Builhe C, Kissa K, Kremer EJ (2001) Preferential transduction of neurons by canine adenovirus vectors and their efficient retrograde transport in vivo. FASEB J 15:2283–2285

    PubMed  CAS  Google Scholar 

  5. Peltekian E, Garcia L, Danos O (2002) Neurotropism and retrograde axonal transport of a canine adenoviral vector: a tool for targeting key structures undergoing neurodegenerative processes. Mol Ther 5:25–32

    Article  PubMed  CAS  Google Scholar 

  6. Alcayaga-Miranda F, Cascallo M, Rojas JJ, Pastor J, Alemany R (2010) Osteosarcoma cells as carriers to allow antitumor activity of canine oncolytic adenovirus in the presence of neutralizing antibodies. Cancer Gene Ther 17:792–802

    Article  PubMed  CAS  Google Scholar 

  7. Smith BF, Curiel DT, Ternovoi VV, Borovjagin AV, Baker HJ, Cox N et al (2006) Administration of a conditionally replicative oncolytic canine adenovirus in normal dogs. Cancer Biother Radiopharm 21:601–606

    Article  PubMed  CAS  Google Scholar 

  8. Hemminki A, Kanerva A, Kremer EJ, Bauerschmitz GJ, Smith BF, Liu B et al (2003) A canine conditionally replicating adenovirus for evaluating oncolytic virotherapy in a syngeneic animal model. Mol Ther 7:163–173

    Article  PubMed  CAS  Google Scholar 

  9. Bouet-Cararo C, Contreras V, Fournier A, Jallet C, Guibert JM, Dubois E et al (2011) Canine adenoviruses elicit both humoral and cell-mediated immune responses against rabies following immunisation of sheep. Vaccine 29:1304–1310

    Article  PubMed  CAS  Google Scholar 

  10. Henderson H, Jackson F, Bean K, Panasuk B, Niezgoda M, Slate D et al (2009) Oral immunization of raccoons and skunks with a canine adenovirus recombinant rabies vaccine. Vaccine 27:7194–7197

    Article  PubMed  CAS  Google Scholar 

  11. Hu R, Zhang S, Fooks AR, Yuan H, Liu Y, Li H et al (2006) Prevention of rabies virus infection in dogs by a recombinant canine adenovirus type-2 encoding the rabies virus glycoprotein. Microbes Infect 8:1090–1097

    Article  PubMed  CAS  Google Scholar 

  12. Hu RL, Liu Y, Zhang SF, Zhang F, Fooks AR (2007) Experimental immunization of cats with a recombinant rabies-canine adenovirus vaccine elicits a long-lasting neutralizing antibody response against rabies. Vaccine 25:5301–5307

    Article  PubMed  CAS  Google Scholar 

  13. Liu Y, Zhang S, Ma G, Zhang F, Hu R (2008) Efficacy and safety of a live canine adenovirus-vectored rabies virus vaccine in swine. Vaccine 26:5368–5372

    Article  PubMed  CAS  Google Scholar 

  14. Dormond E, Perrier M, Kamen A (2009) From the first to the third generation adenoviral vector: what parameters are governing the production yield? Biotechnol Adv 27:133–144

    Article  PubMed  CAS  Google Scholar 

  15. Fernandes P, Peixoto C, Santiago VM, Kremer EJ, Coroadinha AS, Alves PM (2013) Bioprocess development for canine adenovirus type 2 vectors. Gene Ther 20(4):353–360

    Article  PubMed  CAS  Google Scholar 

  16. Segura MM, Puig M, Monfar M, Chillon M (2012) Chromatography purification of canine adenoviral vectors. Hum Gene Ther Methods 23(3):182–197

    Article  PubMed  CAS  Google Scholar 

  17. Altaras NE, Aunins JG, Evans RK, Kamen A, Konz JO, Wolf JJ (2005) Production and formulation of adenovirus vectors. Adv Biochem Eng Biotechnol 99:193–260

    PubMed  CAS  Google Scholar 

  18. Segura MM, Kamen AA, Garnier A (2011) Overview of current scalable methods for purification of viral vectors. Methods Mol Biol 737:89–116

    Article  PubMed  CAS  Google Scholar 

  19. Segura MM, Monfar M, Puig M, Mennechet F, Ibanes S, Chillon M (2010) A real-time PCR assay for quantification of canine adenoviral vectors. J Virol Methods 163:129–136

    Article  PubMed  CAS  Google Scholar 

  20. Tancevski I, Wehinger A, Patsch JR, Ritsch A (2006) In vivo application of adenoviral vectors purified by a Taqman Real Time PCR-supported chromatographic protocol. Int J Biol Macromol 39:77–82

    Article  PubMed  CAS  Google Scholar 

  21. Hutchins B (2002) Development of a reference material for characterizing adenovirus vectors. BioProcess J 1:25–29

    Google Scholar 

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Puig, M., Piedra, J., Miravet, S., Segura, M.M. (2014). Canine Adenovirus Downstream Processing Protocol. In: Chillón, M., Bosch, A. (eds) Adenovirus. Methods in Molecular Biology, vol 1089. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-679-5_14

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  • DOI: https://doi.org/10.1007/978-1-62703-679-5_14

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-678-8

  • Online ISBN: 978-1-62703-679-5

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